Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

Landscape simplification increases vineyard pest outbreaks and insecticide use

Diversifying agricultural landscapes may mitigate biodiversity declines and improve pest management. Yet landscapes are rarely managed to suppress pests, in part because researchers seldom measure key variables related to pest outbreaks and insecticides that drive management decisions. We used a 13‐year government database to analyse landscape effects on European grapevine moth (Lobesia botrana) outbreaks and insecticides across c. 400 Spanish vineyards. At harvest, we found pest outbreaks increased four‐fold in simplified, vineyard‐dominated landscapes compared to complex landscapes in which vineyards are surrounded by semi‐natural habitats. Similarly, insecticide applications doubled in vineyard‐dominated landscapes but declined in vineyards surrounded by shrubland. Importantly, pest population stochasticity would have masked these large effects if numbers of study sites and years were reduced to typical levels in landscape pest‐control studies. Our results suggest increasing landscape complexity may mitigate pest populations and insecticide applications. Habitat conservation represents an economically and environmentally sound approach for achieving sustainable grape production.     

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus–oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL⁺) and without (CCL^-) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL⁺), contralateral (CL⁻), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL⁻ cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL⁺ group; however, a higher proportion of embryos were obtained from the CCL⁻ group. Besides, COCs from the CL⁺ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL⁻ group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.     

A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins

Rationale: The αvβ6- and αvβ8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFβ complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called “5a”), which selectively recognizes the LAP/TGFβ complex-binding site of αvβ6 and αvβ8.Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with ¹⁸F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvβ6/αvβ8 integrins and various αvβ6/αvβ8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvβ8-positive prostate tumors.Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFβ activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvβ6/αvβ8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvβ6/αvβ8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvβ8-positive prostate tumors.Conclusions: The results indicate that 5a can home to αvβ6- and/or αvβ8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvβ6/αvβ8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFβ activators.     

Human salivary gland branching morphogenesis: morphological localization of claudins and its parallel relation with developmental stages revealed by expression of cytoskeleton and secretion markers

Development of salivary glands is a highly complex and dynamic process termed branching morphogenesis, where branched structures differentiate into mature glands. Tight junctions (TJ) are thought to play critical roles in physiological functions of tubular organs, contributing to cell polarity and preventing lateral movement of membrane proteins. Evidence demonstrated that claudins are directly involved in TJ formation and function. Using immunohistochemistry and immunofluorescence we have mapped the distribution of claudins-1, 2, 3, 4, 5, 7 and 11 and compared it with the expression of differentiation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. Expression of all claudins, except claudin-2 was detected in the various phases of human salivary gland development, up to fully mature salivary gland. The expression of all claudins increased according to the progression of salivary gland maturation evidenced by the classical markers-cytokeratin 14, cytokeratin low molecular weight, smooth muscle actin and human secretory component. Tight junction proteins-claudins appear to be important in the final shape and physiological functions of human salivary glands and are parallel related with markers of salivary gland differentiation.     

The 5.5-year results of MegaOATS – autologous transfer of the posterior femoral condyle: a case-series study

Introduction

Large osteochondral defects of the weight-bearing zones of femoral condyles in young and active patients were treated by autologous transfer of the posterior femoral condyle (large osteochondral autogenous transplantation system (MegaOATS)). The technique presented is a sound and feasible salvage procedure to address large osteochondral defects in weight-bearing zones.

Methods

Thirty-six patients between July 1996 and December 2000 were included. Thirty-three patients (10 females, 23 males) were evaluated by the Lysholm score and X-ray scans. A random sample of 16 individuals underwent magnetic resonance imaging analysis. The average age at the date of surgery was 34.3 (15 to 59) years, and the mean follow up was 66.4 (46 to 98) months. The mean defect size was 6.2 (2 to 10.5) cm², in 27 patients affecting the medial femoral condyle and in six patients affecting the lateral femoral condyle. Trauma or osteochondrosis dissecans were pathogenetic in 82%.

Results

The Lysholm score in all 33 individuals showed a highly significant increase from a preoperative median 49.0 points to a median 86.0 points (P ≤ 0.001). Twenty-seven patients returned to recreational sports. X-ray scans showed a rounding of the osteotomy edge in 24 patients, interpreted as a partial remodelling of the posterior femoral condyle. Preoperative osteoarthritis in 17 individuals was related to significant lower Lysholm scores (P = 0.014), but progression in 17 patients did not significantly influence the score results (P = 0.143). All 16 magnetic resonance imaging examinations showed vital and congruent grafts.

Conclusion

Patients significantly improve in the Lysholm score, in daily-life activity levels and in return to recreational sports. Thirty-one out of 33 patients were comfortable with the results and would undergo the procedure again. The MegaOATS technique is therefore recommended as a salvage procedure for young individuals with large osteochondral defects in the weight-bearing zone of the femoral condyle.     

Chromosomal diversity in two synchronopatric species of Hoffmannseggella (Orchidaceae)

Hoffmannseggella viridiflora Verola & Semir (Laeliinae, Orchidaceae) is a recently discovered species in the campos rupestres vegetation of the Espinhaço Range, MG, Brazil, in synchronopatry with H. bradei (Pabst) V. P. Castro & Chiron. Both morphological and phenological studies indicate that these species are closely related. To substantiate the differentiation of these two species we examined their chromosome numbers and morphologies. The two species had different chromosome numbers, with H. bradei having 2n = 40 and H. viridiflora having 2n = 44 chromosomes, an aneuploid number not previously documented in the genus. Meiotic behavioral studies undertaken with H. bradei revealed many abnormalities related to bivalent numbers and chromosome migration, suggesting that meiotic abnormalities could generate aneuploid gametes and perhaps aneuploid zygotes. Karyotype formulas and chromosome morphologies are quite different between the species, so H. viridiflora was not directly derived from H. bradei through simple chromosome additions. Complementary analyses are necessary to understand the process and species involved in the origin of H. viridiflora.     

Burial of nonpolar surface area and thermodynamic stabilization of globins as a function of chain elongation

Proteins are biosynthesized from N to C terminus before they depart from the ribosome and reach their bioactive state in the cell. At present, very little is known about the evolution of conformation and the free energy of the nascent protein with chain elongation. These parameters critically affect the extent of folding during ribosome‐assisted biosynthesis. Here, we address the impact of vectorial amino acid addition on the burial of nonpolar surface area and on the free energy of native‐like structure formation in the absence of the ribosomal machinery. We focus on computational predictions on proteins bearing the globin fold, which is known to encompass the 3/3, 2/2, and archaeal subclasses. We find that the burial of nonpolar surface increases progressively with chain elongation, leading to native‐like conformations upon addition of the last C‐terminal residues, corresponding to incorporation of the last two helices. Additionally, the predicted folding entropy for generating native‐like structures becomes less unfavorable at nearly complete chain lengths, suggesting a link between the late burial of nonpolar surface and water release. Finally, the predicted folding free energy takes a progressive favorable dip toward more negative values, as the chain gets longer. These results suggest that thermodynamic stabilization of the native structure of newly synthesized globins during translation in the cell is significantly enhanced as the chain elongates. This is especially true upon departure of the last C‐terminal residues from the ribosomal tunnel, which hosts ca., 30–40 amino acids. Hence, we propose that release from the ribosome is a crucial step in the life of single‐domain proteins in the cell. Proteins 2014; 82:2318–2331. © 2014 Wiley Periodicals, Inc.